WO2002000697A2 - Gene bonsai, proteine de liaison de phospholipide, necessaire pour assurer la tolerance a la chaleur dans des plantes de la famille arabidopsis - Google Patents

Gene bonsai, proteine de liaison de phospholipide, necessaire pour assurer la tolerance a la chaleur dans des plantes de la famille arabidopsis Download PDF

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WO2002000697A2
WO2002000697A2 PCT/US2001/020172 US0120172W WO0200697A2 WO 2002000697 A2 WO2002000697 A2 WO 2002000697A2 US 0120172 W US0120172 W US 0120172W WO 0200697 A2 WO0200697 A2 WO 0200697A2
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plant
bonl
nucleic acid
transgenic plant
transgenic
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PCT/US2001/020172
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WO2002000697A3 (fr
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Jian Hua
Paula Grisafi
Gerald R. Fink
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Whitehead Institute For Biomedical Research
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Publication of WO2002000697A3 publication Critical patent/WO2002000697A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • Multicellular organisms develop and maintain a relatively constant size and shape over a wide range of different environmental conditions. This homeostasis is accomplished both by extrinsic mechanisms, e.g., movement that can reestablish a constant environment, and also by intrinsic mechanisms, e.g., mechanisms that alter cellular metabolism so that the organism can retain its morphology despite the altered environment. Animals have the ability to relocate themselves to a different location to. aintain constant external conditions, whereas most plants are anchored and can only bend in response to changes in environmental factors such as light, gravity, or mechanical force.
  • extrinsic mechanisms e.g., movement that can reestablish a constant environment
  • intrinsic mechanisms e.g., mechanisms that alter cellular metabolism so that the organism can retain its morphology despite the altered environment.
  • Animals have the ability to relocate themselves to a different location to. aintain constant external conditions, whereas most plants are anchored and can only bend in response to changes in environmental factors such as light,
  • Wild-type Arabidopsis plants maintain a relatively constant size over a wide range of temperatures.
  • This homeostasis requires the BONZAI1 ⁇ BONl) gene because bonl null mutants produce miniature fertile plants at 22°C, but produce a wild-type phenotype when grown at 28°C.
  • the expression of BONl and a BON1- associated protein ⁇ BAPl) is modulated by temperature.
  • BONl and BAPl have a direct role in regulating cell expansion and cell division at lower temperatures.
  • BONl contains a Ca -dependent phospholipid-binding domain and is associated with the plasma membrane. It belongs to the copine gene family, which is conserved from protozoa to humans. This gene family may function in the pathway of membrane trafficking in response to external conditions.
  • BONZAI1 ⁇ BONl), BON2, and BON3 genes permit wild-type Arabidopsis plants to maintain a relatively constant size over a wide range of temperatures, bonl null mutants produce miniature fertile plants at 22°C, but produce a wild-type phenotype when grown at 28°C.
  • a protein associated with BONl, "BONl -associated protein", or BAPl is also modulated by temperature.
  • BONl and BAPl have a direct role in regulating cell expansion and cell division at temperatures lower than those at which Arabidopsis is normally grown.
  • BONl contains a Ca -dependent phospholipid-binding domain and is associated with the plasma membrane, and belongs to the copine gene family, which is conserved from protozoa to humans.
  • the present invention features an isolated nucleic acid comprising SEQ ID NO:2, and degenerate variants thereof.
  • the invention also features an isolated nucleic acid comprising 315 or more consecutive nucleotides of SEQ ID NO:2, and also an isolated nucleic acid comprising a sequence that encodes a polypeptide having an amino acid sequence which is 94% identical to SEQ ID NQ:3..._ Theinvention also_f eatures..an-isolated-polypeptide-comprising-the -amino- acid sequence of SEQ ID NO:3, and also an isolated polypeptide comprising 400 or more consecutive amino acid residues of SEQ ID NO:3, and also an isolated polypeptide comprising a sequence that has 94% sequence identity to SEQ ID NO:3.
  • the present invention features a vector comprising the isolated nucleic acids of SEQ ID NO: 2 or degenerate variants thereof, and isolated cells ⁇ e.g., a prokaryotic cell, a eukaryotic cell) containing such a vector.
  • the present invention features a transgenic -plant which has an altered size compared to a conesponding non-transgenic plant, comprising introducing into the plant exogenous nucleic acid which modulates BONl in the plant.
  • the transgenic plant can be smaller in size.
  • the invention features a transgenic plant that is smaller in size than a conesponding non-transgenic plant, comprising introducing exogenous nucleic acid which inhibits BONl in the plant.
  • the exogenous nucleic acid can be inserted in the twelfth exon of the BONl gene.
  • the exogenous nucleic acid can be a fusion of BONl with a beta-glucuronidase gene.
  • the exogenous nucleic acid can be inserted in the second exon of the BONl gene.
  • the invention also features a transgenic tissue culture derived from the transgenic plant as described above, and a transgenic seed of such a transgenic plant, and a transgenic plant, plant part, plant cell or tissue culture, grown from such a transgenic seed.
  • the transgenic plant, plant part, plant cell or tissue culture can be an ornamental plant or a rurfgrass, or the plant part, plant cell or tissue culture can be derived from an ornamental plant or a rurfgrass.
  • the invention features a transgenic plant that is larger in size than a conesponding non-transgenic plant, comprising introducing into the plant an exogenous nucleic acid which enhances BONl in the plant.
  • the the exogenous nucleic acid can be an exogenous BONl gene.
  • the invention also features a method of producing a transgenic plant which has an altered size compared to a conesponding non-transgenic plant, comprising introducing into the plant exogenous nucleic acid which modulates BONl in the plant..
  • the invention also features a method of producing a transgenic plant that is smaller in size than a conesponding non-transgenic plant, comprising introducing into the plant an exogenous nucleic acid which inhibits BONl in the plant.
  • the method can be accomplished by mutating the endogenous BONl in the plant.
  • the exogenous nucleic acid can be a fusion of BONl with a beta-glucuronidase gene (BON1-GUS).
  • BON1-GUS beta-glucuronidase gene
  • the exogenous nucleic acid can result in overexpression of the C- terminus of a BONl, or overexpression of the full length BONl.
  • the transgenic plant can be an angiosperm or a gymnosperm.
  • the transgenic plant can be an ornamental plant or a turfgrass.
  • the invention also features a method of producing a transgenic plant that is larger in size than a conesponding non-transgenic plant, comprising introducing into the plant an exogenous nucleic acid which enhances BONl in the plant.
  • the exogenous nucleic acid can be an exogenous BONl gene.
  • the plant can be a crop plant, or a plant grown and harvested for its biomass.
  • the invention also features a method of modulating homeostasis of a plant, the method comprising introducing into the plant an exogenous nucleic acid which modulates BONl in the plant.
  • the present invention relates to a method to increase the yield of a plant, comprising introducing an exogenous nucleic acid that enhances BONl in the plant.
  • the method can be used to produce a plant that can grow at a higher altitude or in a lower temperature region than a conesponding non-transgenic plant, where the method comprises introducing into the plant an exogenous nucleic acid that enhances BONl in the plant.
  • the invention features an isolated nucleic acid comprising SEQ JJD NO:5, and degenerate variants thereof.
  • the nucleic acid can comprise 170 or more consecutive nucleotides of SEQ ID NO:5.
  • the nucleic acid can comprise a sequence that encodes a polypeptide having an amino acid sequence which is 97% identical to SEQ ID NO:6.
  • the invention also features an isolated polypeptide comprising the amino acid sequence of SEQ JJJ NO:6, and an isolated polypeptide comprising 550 or more consecutive arnino_acid residues of SEQ-ID.N :6,-an anisQlated.polypeptide comprising a sequence that has 97% sequence identity to SEQ ID NO:6.
  • the present invention features a vector comprising the isolated nucleic acids of SEQ ID NO: 5 or degenerate variants thereof, and isolated cells ⁇ e.g., a prokaryotic cell, a eukaryotic cell) containing such a vector.
  • the invention features an isolated nucleic acid comprising SEQ ID NO:l 1, and degenerate variants thereof.
  • the nucleic acid can comprise 390 or more consecutive nucleotides of SEQ ID NO: 11.
  • the nucleic acid can comprise a sequence having 99% sequence identity to the coding sequence of SEQ ID NO: 11.
  • the nucleic acid can comprise a sequence that encodes a polypeptide having an amino acid sequence which is 98% identical to SEQ ID NO:12.
  • the invention also features an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 12, and an isolated polypeptide comprising 180 or more consecutive amino acid residues of SEQ ID NO: 12, and an isolated polypeptide comprising a sequence that has 97% sequence identity to SEQ ID NO: 12.
  • the present invention features a vector comprising the isolated nucleic acids of SEQ ID NO: 11 or degenerate variants thereof, and isolated cells ⁇ e.g., a prokaryotic cell, a eukaryotic cell) containing such a vector.
  • the present invention features a method of producing a transgenic plant which has an altered size compared to a conesponding non- transgenic plant, comprising introducing into the plant exogenous nucleic acid which modulates BAPl in the plant.
  • the method is a method of producing a transgenic plant that is smaller in size than a conesponding non- transgenic plant comprising introducing into the plant an exogenous nucleic acid sequence which inhibits BAPl.
  • the method can be accomplished by mutating the endogenous BAPl in the plant.
  • the transgenic plant can be an angiosperm or a gymnosperm.
  • the transgenic plant can be an ornamental plant or a turfgrass.
  • the invention also features a transgenic tissue culture derived from the transgenic plant as described above, and a transgenic seed of such a transgenic plant,..and-a.t ⁇ ansgenic plant, ..plant, part, plantxell or tissuexulture,_grown from such a transgenic seed.
  • the transgenic plant, plant part, plant cell or tissue culture can be an ornamental plant or a turfgrass, or the plant part, plant cell or tissue culture can be derived from an ornamental plant or a turfgrass.
  • the invention also features a method of producing a transgenic plant that is larger in size than a conesponding non-transgenic plant, comprising introducing into the plant an exogenous nucleic acid which enhances BAPl in the plant.
  • the exogenous nucleic acid can be an exogenous BAPl gene.
  • the plant can be a crop plant, or a plant grown and harvested for its biomass.
  • the invention also features a method of modulating homeostasis of a plant, the method comprising introducing into the plant an exogenous nucleic acid which modulates BAPl in the plant.
  • the method can be used to increase the yield of a plant.
  • the method can be used to produce a plant that can grow at a higher altitude or in a lower temperature region than a conesponding non-transgenic plant, where the method comprises introducing into the plant an isolated nucleic acid that enhances BAPl in the plant.
  • the invention also features a transgenic plant produced by any of the methods described herein.
  • Figs 1A, IB, 1C, ID, IE and IF are a set of six photographs showing the phenotype of bonl-1 plants.
  • Fig. 1 A shows wild type Arabidopsis (Col-0) and bonl-1 grown at 22°C.
  • Fig. IB shows wild type Arabidopsis (Col-0) and bonl-1 grown at 28°C.
  • Fig. 1C shows leaves of wild type Arabidopsis (Col-0) and bonl-1 grown at 22°C.
  • Fig. ID shows stems of wild type Arabidopsis (Col-0) and bonl-1 grown at 22°C.
  • Fig. IE shows wild type Arabidopsis (Col-0) and bonl-1 grown at 22°C for three weeks and then transferred to 28°C for ten days.
  • Fig. IF shows wild type Arabidopsis (Col-0) and bonl-1 grown at 28°C for three weeks and then transfened to 22°C for ten days.
  • Figs 2 A and 2B are a set of two diagrams illustrating the structure of the ⁇ -ONJ-gene-(Fig..2 A)-andihe BOM structure of the BONl gene. Exons are represented by boxes, and translation start and stop are indicated by "ATG” and "STOP", respectively. The T-DNA insertion sites in bonl-1 and bonl-2 are in exons 12 and 2 as indicated.
  • Fig. 2B shows the domains of the BONl protein, which has two C2 domains at the N-terminus and an
  • Fig. 3 is a diagram showing the alignment of the amino acid sequences for
  • Arabidopsis thaliana BONl (SEQ ID NO:3), BON2 (SEQ ID NO:6) and BON3 (SEQ ID NO:9) proteins, aligned with the COPINEl protein (SEQ ID NO: 16) from
  • Figs. 4A, 4B, 4C and 4D show BONl expression.
  • Figs. 4A, 4B and 4C are a set of three photographs showing the BON1-GUS expression pattern. Dark blue staining indicates GUS expression.
  • Fig. 4A shows expression in seedlings at the 4- leaf stage; expression is seen in young leaves.
  • Fig. 4B- shows expression in seedlings before bolting; expression is seen in younger leaves and not in older ones.
  • Fig. 4C shows expression in the inflorescence stem; expression is seen in the apical portion of the stem.
  • Fig. 4D is a Northern blot analysis showing that BONl RNA expression is modulated by temperature. RNA samples were isolated from 5-week-old plants grown at 28°C (lane 1), shifted from 28°C to 22°C for 12 hours
  • Figs. 5A, 5B, 5C and 5D show the association of BONl with membranes.
  • Figs. 5A, 5B, 5C and 5D are photographs of SDS-PAGE gels stained with
  • Fig. 5A shows that BONl binds lipid.
  • BONl protein was mixed with (+) or without (-) phosphotidyl serine (PS) or calcium ion. The mixture was spun down and the protein in the pellet was run on SDS-PAGE and stained with
  • Fig. 5B shows that BONl is associated with membranes in the plants.
  • Total proteins from BON1-HA plants were extracted and incubated with buffer, buffer with 0.5 M NaCl, 2.5 M urea, 0.1 M Na 2 CO 3 , 1% Triton X-l 00, and 1% sarcosyl, respectively, for one hour.
  • the soluble and pellet fractions were separated by ultracentrifugation.
  • Fig. 5C shows that calcium stimulates the association of BONl with membranes.
  • Fig. 5D shows subcellular fractionation by sucrose density centrifugation.
  • the microsomal fraction from transgenic Arabidopsis containing BONl -HA was separated on a sucrose gradient of 25% to 50%. Proteins from each fraction were separated on a 4- 20% SDS-PAGE gel and blotted to membranes.
  • blots were probed with anti- HA antibody, and antibodies against marker proteins from different membranes: plasma-membrane ATPase (PM-ATPase) for plasma membrane, BIP for endoplasmic reticulum, pyrophosphate (PPase) for vacoules.
  • PM-ATPase plasma-membrane ATPase
  • BIP endoplasmic reticulum
  • PPase pyrophosphate
  • the activity of Golgi specific UDPase activity was assayed (Schaller, G.E. and N.D. DeWitt (1995) Methods Cell Biol. 50:129-48) in each fraction and the strength of the activity was indicated from very strong ( M M ) to low (-).
  • Fig. 6 is a bar graph showing vesicle aggregation promoted by BONl in vitro.
  • Vesicle aggregation was monitored by the turbidity of the lipid, which was measured by the absorbance at 540 nm.
  • Phosphotidylserine was incubated with no protein ("A"), control protein ("B"), the two C2 domains of BONl (C2A-C2B) ("C"), or full-length BONl protein (C2A-C2B-A) ("D") in the presence (Ca 2+ ) or absence (-) of calcium ion for one-half hour.
  • Figs. 7A and 7B are a pair of Northern blots showing BAPl expression and function.
  • Fig. 7C is a photograph showing suppression of the bonl-1 phenotype by BAPl overexpression.
  • Fig. 7A is a Northern blot of total RNAs from different tissues (root, leaf, stem, and flower), showing tissue distribution of BAPl. BONl and BAPl show higher expression in leaves and stems and BAPl also has higher expression in roots.
  • Fig. 7B shows BAPl RNA expression in wild type and the bonl-1 mutant grown at 22°C and 28°C.
  • Fig. 7C shows suppression of the bonl-1 phenotype by BAPl overexpression. Wild type and bonl-1 axe on the left for comparison.
  • Four independent transgenic lines of 35 S::BAP1 in a bonl-1 background are on the right. The plants also show varying degrees of suppression of bonl-1 phenotype by overexpression of BAPl.
  • Figs. 9A and 9B respectively, show the BONl coding (SEQ ID NO:5) and BON2 protein (SEQ ID NO:6) sequences.
  • Figs. 10A and 10B respectively, show the B0N3 coding (SEQ ID NO:8) and BON3 protein (SEQ ID NO:9) sequences.
  • Figs. 11 A and 1 IB show the BAPl coding (SEQ ID NO: 11) and BAPl protein (SEQ ID NO: 12) sequences.
  • Figs. 12A and 12B respectively, show the BAL coding (SEQ ID NO: 14) and
  • BAL protein (SEQ ID NO: 15) sequences.
  • the invention relates to nucleic acids (DNA, cDNA, RNA, mRNA) and proteins which modulate plant growth homeostasis.
  • the nucleic acids and proteins described herein control cell expansion and cell division, resulting in changes in the size and rate at which the host plant grows when exposed to lower temperatures.
  • the present invention relates to nucleic acids expresing the BONZAI1 protein ⁇ BONl), and its homo logs BON2 and BON3, the .gONZ-associated protein ⁇ BAPl) and the BAL :BAP-Like protein ⁇ BAL).
  • the Arabidopsis BONl genomic sequence is presented as SEQ ID NO: 1, and the cDNA as SEQ JD NO:2.
  • the predicted BONl protein sequence is presented as SEQ ID NO:3. Two homologs of the BONl protein, BON2 and BON3 are also disclosed herein.
  • the BON2 genomic sequence is presented as SEQ JD NO:4, the BON2 coding sequence as SEQ ID NO:5, and the predicted BON2 protein sequence as SEQ ID NO:6.
  • the BON3 genomic sequence is presented as SEQ ID NO:7, the BON3 coding sequence as SEQ ID NO:8, and the predicted BON2 protein sequence as SEQ ID NO:9.
  • the Arabidopsis BAPl genomic sequence is presented as SEQ ID NO: 10, and the BAPl coding sequence as SEQ ID NO: 11.
  • the predicted BAPl protein sequence is presented as SEQ ID NO: 12.
  • the BAL genomic sequence is presented as SEQ ID NO: 13
  • the BAL coding sequence as SEQ ID NO: 14.
  • the predicted BAL protein sequence is presented as SEQ ID NO : 15.
  • One approach to identifying the genes involved in temperature homeostasis is to isolate Arabidopsis mutants that are unable to maintain size or shape over a broad temperature range. A mutant was isolated using this approach, bonzail ⁇ bon ⁇ ), which makes miniature plants at lower temperatures.
  • the BONl gene encodes one of the copines, a protein widely conserved in plants and animals.
  • the BONl protein is tightly associated with the plasma membrane and promotes aggregation of lipid vesicles in vitro.
  • BONl associates with another protein BAPl which, when overexpressed, can suppress the bonl phenotype.
  • the copine gene family may function in membrane trafficking and be transcriptionally regulated by the environmental conditions to which they are designed to respond.
  • the invention is directed to isolated nucleic acids, BONl, BON2, BON3, BAPl and BAL, which encode proteins that are necessary for normal growth, hihibition of one or more of these genes results in plants which are smaller in size (minaturized) when grown at lower temperature, compared to the size of the conesponding wild type plant (the same type of plant as the mutant but which does not have a defect in one or more of these genes) when grown at the same lower temperature. Enhancement of one or more of these genes results in plants which are larger in size, compared to the size of the conesponding wild type plant (the same type of plant as the mutant but which does not have a defect in one or more of these genes).
  • the present invention also relates to transgenic plants having altered size (smaller in size, larger in size) compared to the size of a conesponding wild type plant, wherein the transgenic plant comprises exogenous nucleic acid which modulates (inhibits, enhances) BONl , BON2, BON3, BAP 1 and/or BAL in the transgenic plant.
  • exogenous nucleic acid is nucleic acid which is obtained from_a sonrce.other.than the recipienLplan celL(z.e:, the.plant cell, into which the exogenous nucleic acid is being introduced), and which, when encoding a peptide, is stably expressed.
  • the exogenous nucleic acid can be present episomally or integrated into the genome of the plant cell (into genomic nucleic acid).
  • the exogenous nucleic acid can be DNA, DNA obtained from a source in which it occurs in nature, or produced by synthetic or recombinant methods.
  • the present invention relates to a transgenic plant that is smaller in size than a conesponding non-transgenic plant, wherein the plant comprises exogenous nucleic acid which inlribits BONl, BON2, BON3 and/or BAPl in the plant.
  • the inhibition of BONl, BON2, BON3 or BAPl can be partial or complete. Because BONl, BON2, BON3 and BAPl are likely involved in a physiological pathway, inhibiting BONl, BON2, BON3 or BAPl includes, for example, inhibition of the gene by knocking out the gene or mutating the gene in a way to render it non-functional ⁇ e.g., so that the gene does not encode a functional protein).
  • Inhibition also includes interfering with the regulatory region of the gene, e.g., introducing a nucleic acid which prevents expression or the timing of the encoded product ⁇ e.g., splicing in a regulatory region, either upstream or downstream of the gene, which negatively regulates expression of the gene (interefering with, knocking out, or mutating regulatory sequences ⁇ e.g., promoter, enhancer) associated with the gene).
  • a nucleic acid which prevents expression or the timing of the encoded product e.g., splicing in a regulatory region, either upstream or downstream of the gene, which negatively regulates expression of the gene (interefering with, knocking out, or mutating regulatory sequences ⁇ e.g., promoter, enhancer) associated with the gene).
  • Inhibition also includes inhibition of the gene product ⁇ e.g. , protein, peptide), e.g., preventing the use by the plant of the gene's expression product, by mutating the gene product so that the gene product no longer retains its biological function or its biological function is significantly diminished; mutating a molecule with which the gene product interacts; introducing a molecule ⁇ e.g., peptide, small molecule) that binds the gene product thereby inl ibiting its function.
  • Inhibition also includes inhibiting a gene or gene product upstream in the pathway in which the particular gene is involved, e.g., a gene in the BONl pathway which, when inhibited, results in inhibition of BONl downstream.
  • One method of inhibiting expression of an endogenous gene is cosuppression.
  • --T s has-been-shownin-petuniawhere-introduction-of a recombinant chalcone synthase or dihydroflavonol4-reductase gene suppressed the homologous native genes (Napoli, C. et ⁇ l, 1990, The Plant Cell 2:279-289; van der Krol, A.R. et al, 1990, The Plant Cell 2:291-299), and in tobacco, where transformation of a partial nopaline synthase gene into the plant suppresses the expression of the endogenous conesponding gene (Goring, D.R. et al, 1991, Proc. Nail.
  • expression of a truncated form of the relevant gene in the "sense" orientation can be used to suppress the endogenous expression of the native gene, thus lowering the level of the gene product.
  • Another method of inhibiting expression of a gene is supression or inhibition using antisense techniques.
  • antisense RNA forms double- stranded RNA with a target gene product, thereby inhibiting action by that gene product. This is generally done by linking, in reverse orientation, the nucleic acid encoding the target gene product, downstream of its promoter, into a vector.
  • the nucleic acid encoding the antisense product need not represent the entire gene, nor does it need to be fully homologous to the target RNA, rather, the antisense nucleic acid need only encode an RNA having enough homology to bind to the target RNA, or to be of sufficient length to bind to the target RNA in a region critical to its activity.
  • a transgenic plant that is smaller in size than a conesponding wild type plant is produced by introducing a chimeric fusion protein, such as aBONl-GUS fusion protein (or a£ON2-GUS or BON3-GUS fusion protein) into the plant ⁇ e.g., into the second exon or the twelfth exon of a chimeric fusion protein, such as aBONl-GUS fusion protein (or a£ON2-GUS or BON3-GUS fusion protein) into the plant ⁇ e.g., into the second exon or the twelfth exon of
  • BONl inhibits BONl (or BON2 or BON3).
  • introduction of exogenous nucleic acid which overexpresses of the C-terminus of BONl, BON2 or BON3, or of an N-terminal domain of BAPl can also be used to inhibit the genes.
  • the present invention relates to a transgenic plant that is larger in size than a conesponding non-transgenic plant, wherein the transgenic plant comprises an exogenous nucleic acid which enhances BONl, BON2, BON3 and/or-BAPl in-the-plant.
  • the enhancing of BONl, BON2, BON3 or BAPl can be partial or complete. Because BONl, BON2, BON3 and BAPl are likely involved in a physiological pathway, "enhancing" of BONl, BON2, BON3 or BAPl can include enhancing the gene, including duplicating and/or overexpressing the gene, mutating the gene (full length) in a way that it has increased expression, etc. Enhancing also includes interfering with the regulation of the gene, e.g., enhancing its expression or the tuning thereof, e.g., altering regulatory sequences associated with the gene.
  • Enhancing also includes enhancing the gene product itself, e.g., increasing the ability of the plant to use the product or increasing the activity of the product, either by mutating the gene product so that its use by the plant is increased, or by increasing binding of the expression product to its ligand.
  • Polynucleotides encoding BONl, BON2, BON3, BAPl or BAL can be obtained or isolated from natural sources, recombinantly produced, or chemically synthesized. In one embodiment, polynucleotides encoding BONl , BON2, BON3, BAPl or BAL can be cloned out of isolated DNA or a cDNA library.
  • nucleic acids and polypeptides refened to herein as "isolated" ⁇ e.g., essentially pure) are nucleic acids or polypeptides substantially free ⁇ i.e., separated away from) the material of the biological source from which they were obtained ⁇ e.g., as exists in a mixture of nucleic acids or in cells), and which may have undergone further processing.
  • An isolated nucleic acid is not immediately contiguous with ⁇ i.e., covalently linked to) both of the nucleic acids with which it is immediately contiguous in the naturally- occuning genome of the organism from which the nucleic acid is derived.
  • lower temperature refers to a temperature at which the transgenic plant of the present invention grows smaller in size, compared to a conesponding non-transgenic plant. Such a lower temperature will vary depending upon the particular plant. For example, the normal temperature for a plant which grows in a temperate region ⁇ e.g., Arabidopsis) is from, about 24°C ro about 37°C.
  • Transgenic Arabidopsis in which BONl, BON2, BON3, BAPl and/or BAL are inhibited grows smaller in size at about 22°C, compared to a conesponding non- transgenic Arabidopsis plant at 22°C.
  • a lower temperature for plants -which-grow n a-temperate region is from..about.0°C to .ahout.23.°C,..from..abQut5 C. to about 20°C, and from about 10°C to about 15°C.
  • a lower temperature for a plant is about 22°C.
  • a "lower temperature" will vary depending upon the particular plant and the region ⁇ e.g., tropical, temperate, arctic) in which the wild type plant grown and can be determined by one of ordinary skill in the art.
  • Plants that are "smaller in size” are smaller than the conesponding wild type plant, and refers to their growth during the time of exposure to lower temperatures relative to wild type plants. For instance, as described herein, bonl mutant plants do not grow as large, and produce smaller and fewer cells, when grown at 22°C relative to wild type plants grown at the same temperature. Ordinarily, plants grown at lower temperatures continue to grow at a slower rate, but still produce normal-sized cells. If the bonl plants are grown at 22°C are then moved to exposure to 28°C, however, the new growth of bonl plants grow at the same rate as the wild type plants, with the bonl plants having a portion of older tissue that is "minaturized", and newer tissue that is normally proportioned.
  • the temperature conditions are reversed, that is, if the bonl plants are grown at one temperature ⁇ e.g., 28°C), then switched to lower temperatures ⁇ e.g., 22°C), the bonl plants exhibit older tissue of the same proportions as wild type, while the newer tissues are miniaturized.
  • transgenic plants of the present invention that are larger in size are larger than the conesponding wild type plant.
  • the invention also encompasses alleles, degenerate variants, fragments, mutants, homologs and analogs of BONl, BON2, BON3, BAPl and BAL nucleic acids and proteins.
  • An "allele" of a protein is a polypeptide sequence containing a naturally-occuning sequence variation relative to the polypeptide sequence of the reference polypeptide.
  • allele of a polynucleotide encoding the polypeptide is meant a polynucleotide containing a sequence variation relative to the reference polynucleotide sequence encoding the reference polypeptide, where the allele of the polynucleotide encoding the polypeptide encodes an allelic form of the polypeptide.
  • degenerate variants of a nucleic acid are those variant -nucleic- -aoids-mat-di-f-fer-fiom- the -referenGe-sequenGe due-to the-degenerac oft he- genetic code, and encode a protein having the same amino acid sequence as that protein encoded by the reference nucleic acid. Therefore, a degenerate variant of a BONl, BON2, BON3, BAPl or BAL nucleic acid sequence is a sequence that contains a silent or conservative substitution and therefore encodes the same protein as the reference polynucleotide sequence.
  • a "fragment" of the nucleic acid sequences described herein is a portion of the full length nucleic acid sequence that encodes a protein that retains the biological activity of the full-length protein.
  • Such fragments con comprises, for example, from about 50 to about 2000 nucleotides, from about 100 to about 1500 nucleotides, from about 500 to about 1000 nucleotides.
  • Such nucleic acids can also comprise additional sequences derived from the process of cloning, e.g., amino acid residues or amino acid sequences conesponding to full or partial linker sequences. To be encompassed by the present invention, such fragments, with or without such additional sequences, must have substantially the same biological activity as the natural or full-length version of the reference polypeptide.
  • a “fragment" of a protein is any amino acid sequence shorter than the full length protein, comprising at least about 25 consecutive amino acids of the full length protein. Such a fragment may alternatively comprise about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 consecutive amino acids of the full length protein.
  • the fragment may comprise about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74 or 75 consecutive amino acids of the full polypeptide.
  • the fragments of the proteins described herein can comprise, for example, from about 100 to about 800 amino acids, from about 200 to about 700 amino acids, from about 300 to about 600 amino acids, and from about 500 to about 500 amino acids.
  • the protein fragment can be about 170 consecutive amino acids.
  • the protein fragment can be about 180 consecutive amino acids.
  • the protein fragment can be about 315 consecutive amino acids.
  • the protein fragment can be about 390 consecutive-ammo- acids.—In- another embodiment, the protein -fragment-can-be about 400 consecutive amino acids, h another embodiment, the protein fragment can be about 550 consecutive amino acids.
  • Such molecules can comprise additional amino acids derived from the process of cloning, e.g., amino acid residues or amino acid sequences conesponding to full or partial linker sequences.
  • additional amino acid residues e.g., amino acid residues or amino acid sequences conesponding to full or partial linker sequences.
  • such molecules, with or without such additional amino acid residues must have substantially the same biological activity as the reference polypeptide.
  • proteins that have substantially the same amino acid sequence as BONl, BON2, BON3, BAPl or BAL, or polynucleotides that have substantially the same nucleic acid sequence as the polynucleotides encoding BONl, BON2, BON3, BAPl or BAL.
  • substantially the same sequence means a nucleic acid or polypeptide that exhibits at least about 70 % sequence identity, typically at least about 80% sequence identity with the reference sequence, at least about 90% sequence identity, at least about 95% identity, at least about 97%, at least about 98% sequence identity, or at least about 99% sequence identity with the BONl, BON2, BON3, BAPl or BAL reference sequence.
  • the length of comparison for sequences will generally be at least 75 nucleotide bases or 25 amino acids, more preferably at least 150 nucleotide bases or 50 amino acids, more preferably at least 300 nucleotides or 100 amino acids, and most preferably 600 nucleotides or 200 amino acids.
  • the length for comparisn is the full length nucleic acid or amino acid of BONl, BON2, BON3, BAPl or BAL.
  • Sequence identity refers to the subunit sequence similarity between two polymeric molecules, e.g., two polynucleotides or two polypeptides. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two peptides is occupied by serine, then they are identical at that position.
  • the identity between two sequences is a direct function of the number of matching or identical positions, e.g., if half (e.g., 5 positions in a polymer 10 subunits in length) of the positions in two peptide or compound sequences are identical, then the two sequences are 50% identical; if 90% of the positions, e.g., 9 of 10 are matched, the two sequences share 90% sequence identity i --By ⁇ way-ofexample,.-the amino-acid-sequences-R 2 R 5 R 7 R 10 R 6 R 3 _and.- have 3 of 6 positions in common, and therefore share 50% sequence identity, while the sequences R 2 R 5 R 7 R 10 R 6 R 3 andRgR j R j nR g j have 3 of 5 positions in common, and therefore share 60% sequence identity.
  • sequence identity is a direct function of the number of matching or identical positions. Thus, if a portion of the reference sequence is deleted in a particular peptide, that deleted section is not counted for purposes of calculating sequence identity, e.g., R 2 R 5 R 7 R 10 R 6 R 3 and R 2 R 5 R 7 R 10 R 3 have 5 out of 6 positions in common, and therefore share 83.3% sequence identity. Identity is often measured using sequence analysis software e.g., BLASTN or
  • BLASTP (available at http://www.ncbi.nlm.nih.gov/BLAST/).
  • the present invention also includes fusion proteins and chimeric proteins comprising the BONl, BON2, BON3, BAPl or BAL proteins, their fragments, mutants, homologs, analogs, and allelic variants.
  • a fusion or chimeric protein can be produced as a result of recombinant expression and the cloning process, e.g., the protein can be produced comprising additional amino acids or amino acid sequences conesponding to full or partial linker sequences.
  • the term "fusion or chimeric protein" is intended to encompass changes of this type to the original protein sequence.
  • a fusion or chimeric protein can consist of a multimer of a single protein, e.g., repeats of the proteins, or the fusion and chimeric proteins can be made up of several proteins, e.g., several of the proteins. Such fusion or chimeric proteins can be linked together via post-translational modification ⁇ e.g., chemically linked), or the entire fusion protein may be made recombinantly.
  • the invention also encompasses vectors and host cells comprising the BONl,
  • BON2, BON3, BAPl or BAL nucleic acid sequences comprising culturing the host cells described herein under conditions appropriate to produce BONl , BON2, BON3 , BAP 1 and BAL.
  • vector as used herein means a carrier into which pieces of nucleic acid may be inserted or cloned, which canier functions to transfer the pieces of nucleic acid into a host cell.
  • Such a vector may also bring about the replication and/or expression of the transfened nucleic acid pieces.
  • vectors include nucleic acid molecules derived, e.g., from a plasmid, bacteriophage, or mammalian, plant or insect virus, or non- viral vectors such as ligand-nucleic acid conjugates, liposomes, or lipid-nucleic acid complexes. It may be desirable that the transfened nucleic molecule is operatively linked to an expression control sequence to form an expression vector capable of expressing the transfened nucleic acid.
  • Such transfer of nucleic acids is generally called "transformation,” and refers to the insertion of an exogenous polynucleotide into a host cell, inespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • operably linked refers to a situation wherein the components described are in a relationship permitting them to function in their intended manner, e.g., a control sequence "operably linked” to a coding sequence is ligated in such a manner that expression of the coding sequence is achieved under conditions compatirjle with the control sequence.
  • a "coding sequence” is a polynucleotide sequence which is transcribed into mRNA and translated into a polypeptide when placed under the control of (e.g., operably linked to) appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5 '-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.
  • a polynucleotide Once a polynucleotide has been cloned into a suitable vector, it can be ⁇ transformed-into-an-appropriate-hostcell.
  • host.cell By-"host.cell”is meant a celLwhichhas. been or can be used as the recipient of transfened nucleic acid by means of a vector.
  • Host cells can be prokaryotic or eukaryotic, mammalian,_plant, or insect cells, and can exist as single cells, or as a collection, e.g., as a culture, or in a tissue culture, or in a tissue or an organism.
  • Host cells can also be derived from normal or diseased tissue from a multicellular organism, e.g., a plant.
  • Host cell, as used herein, is intended to include not only the original cell which was transformed with a nucleic acid, but also descendants of such a cell, which still contain the nucleic acid.
  • the isolated polynucleotide encoding one of the proteins of the present invention additionally comprises a polynucleotide linker encoding a peptide.
  • linkers are known to those of skill in the art and, for example the linker can comprise at least one additional codon encoding at least one additional amino acid. Typically the linker comprises one to about twenty or thirty amino acids.
  • the polynucleotide linker is translated, as is the polynucleotide encoding the protein, resulting in the expression of a protein with at least one additional amino acid residue at the amino or carboxyl terminus of the protein.
  • the additional amino acid, or amino acids do not compromise the activity of the protein.
  • the present invention also relates to an isolated nucleic acid comprising a sequence that hybridizes under highly stringent conditions to a coding sequence of the isolated nucleic acid of BONl, BON2, BON3, BAPl or BAL.
  • the present invention also relates to an isolated nucleic acid sequence comprising a sequence that hybridizes under highly stringent conditions to a complement (e.g., fully complementary) of the coding sequence which encodes a BONl, BON2, BON3, BAPl or BAL protein.
  • the polynucleotides and proteins of the present invention can be used to design probes to isolate other proteins, for example, homologs of BON, BON2, BON3, BAPl and BAL. Appropriate hybridization methods are provided in U.S. Pat.
  • oligonucleoti.de probe should preferably follow these parameters: (a) it should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any, and (b) it should be designed to have a T m of approx. 80°C (assuming 2°C for eac A-or T ⁇ and-4°C for each G-or-C)r
  • the oligonucleotide should preferably be labeled with ⁇ - P-ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4 x 10 dpm/pmole.
  • the bacterial culture containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth.
  • Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
  • Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters, followed by lysing, denaturing and baking them.
  • Highly stringent condition are those that are at least as stringent as, for example, lx SSC at 65°C, or lx SSC and 50% formamide at 42°C.
  • Moderate stringency conditions are those that are at least as stringent as 4x SSC at 65°C, or 4x SSC and 50% formamide at 42°C.
  • Reduced stringency conditions are those that are at least as stringent as 4x SSC at 50°C, or 6x SSC and 50% formamide at 40°C.
  • the filter is then preferably incubated at 65 °C for 1 hour with gentle agitation in 6x SSC (20x stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 M EDTA (approximately 10 mL per 150 mm filter).
  • 6x SSC 20x stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH
  • SDS 100 ⁇ g/ml of yeast RNA
  • 10 M EDTA approximately 10 mL per 150 mm filter
  • the filter is then preferably incubated at 65°C with gentle agitation overnigb.tr
  • The- filter is then preferabiy-washedin 500 mL-of-2-x- ⁇ SSC/0:5% ⁇ SDS at- room temperature without agitation, preferably followed by 500 mL of 2x SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
  • a third wash with 0. lx SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional.
  • the filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
  • the positive colonies are then picked, grown in culture, and plasmid DNA isolated using standard procedures.
  • Stringency conditions for hybridization refers to conditions of temperature and buffer composition which permit hybridization of a first nucleic acid sequence to a second nucleic acid sequence, wherein the conditions determine the degree of identity between those sequences which hybridize.to each other. Therefore, "high stringency conditions" are those conditions wherein only nucleic acid sequences which are very similar to each other will hybridize. The sequences may be less similar to each other if they hybridize under moderate stringency conditions. Still less similarity is needed for two sequences to hybridize under low stringency conditions.
  • hybridization conditions By varying the hybridization conditions from a stringency level at which no hybridization occurs, to a level at which hybridization is first observed, conditions can be determined at which a given sequence will hybridize to those sequences that are most similar to it.
  • the precise conditions determining the stringency of a particular hybridization include not only the ionic strength, temperature, and the concentration of destabilizing agents such as formamide, but also on factors such as the length of the nucleic acid sequences, their base composition, the percent of mismatched base pairs between the two sequences, and the frequency of occurrence of subsets of the sequences (e.g., small stretches of repeats) within other non-identical sequences.
  • Washing is the step in which conditions are set so as to determine a minimum level of similarity between the sequences hybridizing with each other. Generally, from the lowest temperature at which only homologous hybridization occurs, a 1% mismatch between two sequences results in a 1°C decrease in the melting temperature (T m ) for any chosen SSC concentration. -Generally, a-doubling ofthe-eoneentration-of SSG-resu-lts-in-an- increase in the T m of about 17°C. Using these guidelines, the washing temperature can be determined empirically, depending on the level of mismatch sought. Hybridization and wash conditions are explained in Current Protocols in Molecular Biology (Ausubel, F.M. et al, eds., John Wiley & Sons, Inc., 1995, with supplemental updates) on pages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
  • the T m in °C (81.5°C + 16.6(log 10 M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L), where "M” is the molarity of monovalent cations ⁇ e.g., Na ), and "L” is the length of the hybrid in base pairs.
  • T m in °C (81.5°C + 16.6(log 10 M) + 0.41(% G + C) - 0.61 (% formamide) - 500/L)
  • M is the molarity of monovalent cations (e.g. , Na + )
  • L is the length of the hybrid in base pairs.
  • the T m in °C (81.5°C + 16.6(log 10 M) + 0.41 (% G + C) - 0.61 (% formamide) - 500/L), where "M” is the molarity of monovalent cations (e.g., Na ), and "L” is the length of the hybrid in base pairs.
  • the present invention also relates to a method of producing a transgenic plant, wherein the size of the plant is altered in comparison to the size of a conesponding wild type plant, comprising introducing into the plant an exogenous nucleic acid that modulates (inhibits, enhances) BONl, BON2, BON3, BAPl, and/or BAL.
  • the invention also relates to plants produced by the methods.
  • the present invention relates to a method of producing a transgenic plant that is smaller in size than a conesponding wild type plant, comprising introducing into the plant an exogenous nucleic acid which inhibits BONl, BON2, BON3, BAPl and/or BAL in the plant.
  • the present invention relates to a method of producing a transgenic plant that is larger in size than a conesponding non-transgenic plant, comprising introducing into the plant an isolated nucleic acid which enhances expression of BONl, BON2, BON3, BAPl and/or BAL protein in the plant.
  • the nucleic acids of the present invention can be used in a variety of ways.
  • the present invention relates to methods of producing plants which are miniature in size, comprising introducing exogenous nucleic acid which inhibits BONl, BON2, BON3, BAPl, or BAL in the plant.
  • the present invention relates to methods of increasing the yield of a plant, comprising-introducing into the plant-an isolated nucleic acid which-enhances- BON1, BON2, BON3, BAPl, or BAL in the plant.
  • the present invention relates to methods of producing a transgenic plant that is able to grow at a higher altitude or in a lower temperature region than a conesponding non- transgenic plant, comprising introducing into the plant an isolated nucleic acid that enhances BONl, BON2, BON3, BAPl, or BAL in the plant.
  • the present invention relates to a method of modulating homeostasis of a plant, the method comprising introducing into the plant an exogenous nucleic acid which modulates BONl in the plant.
  • the present invention also relates to the plants produced by such methods, where the plants are miniature in size, are higher- yielding, grow at higher altitues or in cooler regions, or in which homeostasis regarding temperature and plant growth are modulated by the nucleic acids of the invention.
  • a construct comprising exogenous nucleic acid of the invention, or nucleic acid encoding a functional equivalent as described herein, and a promoter can be incorporated into a vector and introduced into the cell(s) of the plant through methods known and used by those of skill in the art.
  • the nucleic acid to be introduced can be incorporated into a vector, to form a construct, with or without other sequences, or it may be naked nucleic acid, and associated with no such sequences.
  • the construct can also include any other necessary regulators such as terminators or the like, operably linked to the coding sequence.
  • leader sequence such as the untranslated leader from the coat protein mRNA of alfalfa mosaic virus (Jobling, S.A. and Gelirke, L. (1987) Nature 325:622-625) or the maize chlorotic mottle virus (MCMV) leader (Lommel, S.A. et al. (1991) Virology 81:382-385).
  • MCMV maize chlorotic mottle virus
  • Targeting sequences are also useful and can be incorporated into the constructs of this invention.
  • a targeting sequence is usually translated into a peptide which directs the polypeptide product of the coding nucleic acid sequence to a desired location within the cell, such as to the plastid, and becomes separated from the-peptide-after transit-of the peptideis complete or concunently with-transit.
  • Examples of targeting sequences useful in this invention include, but are not limited to, the yeast mitochondrial presequence (Schmitz et al. (1989) Plant Cell 1 :783- 791), the targeting sequence from the pathogenesis-related gene (PR-l) of tobacco (Cornellisen et al. (1986) EMBOJ.
  • vacuole targeting signals Chocuole targeting signals
  • vacuole targeting signals Chocuole targeting signals
  • secretory pathway sequences such as those of the ER or Golgi
  • itraorganellar sequences may also be useful for internal sites, e.g., thylakoids in chloroplasts. Theg, S.M. and Scott, S.V. (1993) Trends in Cell Biol. 3:186-190.
  • a 3' untranslated region (3' UTR) is generally part of the expression plasmid and contains a polyA termination sequence.
  • the termination region which is employed will generally be one of convenience, since termination regions appear to be relatively interchangeable.
  • the octopine synthase and nopaline synthase termination regions derived from the Ti- plasmid of A. tumefaciens, are suitable for such use in the constructs of this invention.
  • the transcriptional initiation region of the construct may provide for constitutive expression or regulated expression, hi addition to the ERAl promoter, many promoters are available which are functional in plants.
  • Constitutive promoters for plant gene expression include, but are not limited to, the octopine synthase, nopaline synthase, or mannopine synthase promoters from Agrobacterium, the cauliflower mosaic virus (35S) promoter, the figwort mosaic vims (FMV) promoter, and the tobacco mosaic vims (TMV) promoter.
  • Constitutive gene expression in plants can also be provided by the glutamine synthase promoter (Edwards et al. (1990) Proc. Natl. Acad. Sci. USA 87:3459-3463), the maize sucrose synthetase 1 promoter (Yang et al. (1990) Proc. Natl. Acad. Sci.
  • Heat-shock promoters can be used for regulated expression of plant genes. Developmentally-regulated, stress-induced, wound-induced or pathogen-induced promoters are also useful.
  • the regulatory region may be responsive to a physical stimulus, such as light, as with the RUBP carboxylase ssu promoter, differentiation signals, or metabolites.
  • the time and level of expression of the sense or antisense orientation can have a definite effect on the phenotype produced. Therefore, the promoters chosen, coupled with the orientation of the exogenous DNA, and site of integration of a vector in the genome, will determine the effect of the introduced gene.
  • regulated promoters also include, but are not limited to, the low temperature Kinl and cor6.6 promoters (Wang et al. (1995) Plant Mol. Biol. 28:605; Wang et al. (1995) Plant Mol. Biol. 28:619-634), the ABA inducible promoter (Marcotte Jr. et al. (1989) Plant Cell 1 :969-976), heat shock promoters, such as the inducible hsp70 heat shock promoter of Drosphilia melanogaster (Freeling, M. et al (1985) Ann. Rev. of Genetics 19: 297-323), the cold inducible promoter from B. napus (White, T.C. et al.
  • the nucleic acid can be introduced into plant cells by a method appropriate to the type of host cells (e.g., transformation, electroporation, transfection, infection).
  • transformation all refer to the introduction of a nucleic acid into a cell by one of the numerous methods known to persons skilled in the art. Transformation of prokaryotic cells, for example, is commonly achieved by treating the cells with calcium chloride so as to render them "competent” to take up exogenous DNA, and then mixing such DNA with the competent cells. Prokaryotic cells can also be infected with a recombinant bacteriophage vector.
  • Nucleic acids can be introduced into cells of higher organisms by viral infection, bacteria-mediated transfer (e.g., Agrobacterium T-DNA delivery system), electroporation, calcium phosphate co-precipitation, microinjection, lipofection, bombardment with nucleic-acid coated particles, mixing with silicon carbide "whiskers", floral dip method or other techniques, depending on the particular cell type.
  • “Introduction”, as used herein, of a nucleic acid into a plant, plant cell, plant part or tissue culture is intended to include both those methods of transformation that are known and used with single-celled organisms (e.g., bacteria, yeast, etc.), and also those methods that are known and used for moving nucleic acid into plants, plant cells, plant parts and tissue cultures.
  • single-celled organisms e.g., bacteria, yeast, etc.
  • microprojectile bombardment see for example, Sanford, J.C. et al, U.S. Patent No. 5,100,792 (1992) can be used.
  • a nucleotide construct or a vector containing the construct is coated onto small particles which are then introduced into the targeted tissue (cells) via high velocity ballistic penetration.
  • the vector can be any vector which permits the expression of the exogenous DNA in plant cells mto which the vector is introduced.
  • the transformed cells are then cultivated under conditions appropriate for the -regeneration of-plants, resultingin production of transgenic plants.
  • nucleic acid constructs of this invention can also be incorporated into specific plant parts through the transformation and transfection techniques described herein.
  • the constructs and methods of this invention can be adapted to any plant part, protoplast, or tissue culture wherein the tissue is derived from a photosynthetic organism.
  • plant part is meant to include a portion of a plant capable of producing a regenerated plant. Preferable plant parts include cells, roots, shoots and meristematic portions thereof.
  • plant parts encompassed by this invention are: leaves, stems, roots, flowers, seeds, epicotyls, hypocotyls, cotyledons, cotyledonary nodes, explants, pollen, ovules, meristematic or embryonic tissue, protoplasts, cells, explants and the like.
  • Transgenic plants can be regenerated from any of these plant parts, including tissue culture or protoplasts, and also from explants. Methods will vary according to the species of plant.
  • nucleic acid constructs include Agrobacterium-mediated transformation (see for example Smith, R.H. et ⁇ l., U.S. Patent No. 5,164,310 (1992)), electroporation (see for example, Calvin, N., U.S. Patent No. 5,098,843 (1992)), introduction using laser beams (see for example, Kasuya, T. et ⁇ , U.S. Patent No. 5,013,660 (1991)) or introduction using agents such as polyethylene glycol (see for example Golds, T. et ⁇ l. (1993) Biotechnology 11 :95-97), and the like.
  • Agrobacterium-mediated transformation see for example Smith, R.H. et ⁇ l., U.S. Patent No. 5,164,310 (1992)
  • electroporation see for example, Calvin, N., U.S. Patent No. 5,098,843 (1992)
  • introduction using laser beams see for example, Kasuya, T. et ⁇ , U
  • plant cells may be transformed with a variety of vectors, such as viral, episomal vectors, Ti plasmid vectors and the like, in accordance with well known procedures.
  • vectors such as viral, episomal vectors, Ti plasmid vectors and the like.
  • the method of introduction of the nucleic acid into the plant cell is not critical to this invention.
  • the constructs of this invention are further manipulated to include genes coding for plant selectable markers.
  • Useful selectable markers include enzymes wliich provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, or the like.
  • enzymes providing- for production of-a-compound-identifiable by- color- change- such as GUS ( ⁇ -glucuronidase), or by luminescence, such as luciferase, are useful.
  • the cells or protoplasts containing the nucleic acids of the invention are obtained, the cells or protoplasts are regenerated into whole plants. Plant cells which have been transformed can be regenerated using known techniques. It is known that practically all plants can be regenerated from cultured cells or tissues. The transformed cells are then cultivated under conditions appropriate for the regeneration of plants, resulting in production of transgenic plants. Choice of methodology for the regeneration step is not critical, with suitable protocols being available for many varieties of plants, tissues and other photosynthetic organisms. See, e.g., Gelvin S.B. and Schilperoort R.A., eds. Plant Molecular Biology Manual, Second Edition, Suppl. 1 (1995) Kluwer Academic Publishers, Boston MA, USA.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • the regenerated plants are transfened to standard soil conditions and cultivated in a conventional manner.
  • plant Because of the plasticity of culture of many plant species, the terms "plant”, “plant part”, “plant cell”, and “tissue culture” are intended to be used somewhat interchangably herein. For instance, it is possible, indeed common in the art of plant -transformation and -tissue culture, -for one ⁇ to-transform either-.aplant,-a plant-part,, one or more plant cells, and/or a tissue culture. Once transformed, any of these may be maintained for a time, converted to another, subcultured, and regenerated into a whole plant, from which seed may then be grown. As an example, one can use microparticle bombardment to transform cells in a single leaf on a plant.
  • the plant may then be grown for a time (or not), and the leaf then separated from the plant, and either maintained on growth medium as a leaf, or (through changing the media components) grown as callus (undifferentiated tissue) or protoplasts (single plant cells).
  • the callus or protoplasts may be either subcultured further as callus or protoplasts, or the callus may be converted to protoplasts, and vice versa.
  • Either may be grown to generate individual plant parts (by manipulation of the hormone conditions in the media to preferentially produce roots and/or shoots, etc.), or to regenerate into whole plants (also by hormone manipulation).
  • the regenerated plant may then again be cultured into plant parts, cuttings, plant cells, callus, tissue cultures, etc., or may be grown for seed.
  • transgenic plant is intended to encompass any subculturing, regenerant, seed, or progeny of the transgenic plant, that canies the introduced nucleic acid. It is also due to this plasticity that the term "derived from” is intended to encompass plants, plant parts, plant cells, tissue cultures and explants that are descended from a given plant. For mstance, in the case where a descendant com plant that has been regenerated from a callus culture which was grown from transformed protoplasts taken from a leaf of an originating maize plant, the descendant plant is said to be "derived from” the originating maize plant. The intervening protoplasts and callus are also said to be "derived from” the originating maize plant.
  • the methods of this invention can be used with in planta or seed transformation techniques which do not require culture or regeneration. Examples of these techniques are described in Bechtold, N. et al. (1993) C.R. Acad. Sci. Paris/Life Sciences 316:118-93; Chang, S.S. et al. ⁇ 1990) Abstracts ofthe Fourth International Conference on Arabidopsis Research, Viemia, p. 28; Feldmann, K.A. and Marks, D.M (1987) Mol. Gen. Genet. 208:1-9; Ledoux, L. et al. (1985)
  • the nucleic acid to be introduced After the nucleic acid to be introduced is stably incorporated into regenerated transgenic plants, it can be transfened to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. The plants are grown and harvested using conventional procedures. Such plants, if they continue to contain the introduced nucleic acid of the invention, are intended to be a "transgenic plant", as the term is used herein.
  • Transgenic plants carrying the construct are examined for the desired phenotype using a variety of methods including, but not limited to, an appropriate phenotypic marker, such as antibiotic resistance or herbicide resistance, or visual observation, or biochemical or other methods of assaying the transgenic plant compared to a conesponding non-transgenic plant.
  • an appropriate phenotypic marker such as antibiotic resistance or herbicide resistance, or visual observation, or biochemical or other methods of assaying the transgenic plant compared to a conesponding non-transgenic plant.
  • conesponding non- transgenic plant is meant a plant of the same species, and if applicable, same cultivar, variety, or genetic background, that was not transformed.
  • transgenic plants include, in one embodiment, transgenic plants which are angiosperms, both monocotyledons and dicotyledons.
  • Transgenic plants include those into which DNA has been introduced and their progeny, produced from seed, vegetative propagation, cell, tissue or protoplast culture, or the like.
  • constructs and methods described herein can be applied to all types of plants and other photosynthetic organisms, including, but not limited to: angiosperms (monocots and dicots), gymnosperms, spore-bearing or vegetatively- reproducing plants and the algae, including the cyanophyta (blue-green algae).
  • Particularly prefened plants are those plants which provide commercially- aluable food crops such as the large and small grains (e.g., com, wheat, rice, sorghum, etc.), oil plants (e.g., canola, sunflower, com, soybean, peanut, etc.), vegetable crops (e.g., luttuce, spinach, pepper, potatoes, tomatoes, broccoli, canots, peas, beans, etc.), fruits (e.g., apple, plum, orange, lemon, etc.), ornamental plants (e.g., rose, cut flowers, etc.), and other commercially-important plants (e.g., cotton, sugar cane, sugar beet- tobacco, grasses-, etc.).
  • the large and small grains e.g., com, wheat, rice, sorghum, etc.
  • oil plants e.g., canola, sunflower, com, soybean, peanut, etc.
  • vegetable crops e.g., luttuce, spinach, pepper
  • Seed can be obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species. Seed may also be obtained from plant parts, e.g., meristems grown in tissue culture. Alternatively, the plant can be vegetatively propagated by culturing plant parts under conditions suitable for the regeneration of such plant parts.
  • the nucleic acid and ammo acid sequences of the present invention can be used in a variety of ways, e.g., the transgenic plants containing the BONl, BON2, BON3, the BAPl and/or the BAL mutant(s) can be used to produce plants which grow slowly at lower temperatures.
  • Such a trait would be useful in turf grasses, for instance, where it maybe undesirable or labor-intensive to maintain, e.g., in low- traffic areas where the appearance of lawn is to be maintained, but the labor of mowing can be reduced.
  • miniaturized plants can be used in situations where a normal-sized plant or plant product is less desirable, e.g., miniaturized ornamental plants, such as smaller flower, vegetable or fruit plants for use by apartment residents, "no-care" bonsai trees, miniature water lilies, miniature fruits as novelties, etc., or low growth where extensive growth is undesirable.
  • miniaturized ornamental plants such as smaller flower, vegetable or fruit plants for use by apartment residents, "no-care" bonsai trees, miniature water lilies, miniature fruits as novelties, etc., or low growth where extensive growth is undesirable.
  • Another embodiment of the invention is the production of "baby vegetables.”
  • Baby corn for instance, is very popular in Asian cuisine, and is the ear or maize which has been harvested young, before pollination has taken place, either before the silks have emerged, or just after, depending on the cultivar. Because baby com is the immature ear of conventional maize, a great deal of land must be used to produce this vegetable, and the plants themselves are close to full height when the ears are produced.
  • bonl corn plants can be made which grow as miniatures at cool temperatures.
  • a relatively southern climate e.g., southern Georgia or northern Florida
  • the plants will grow as miniatures during the cooler (but not freezing) winter.
  • Such com planted for baby co production, can be grown at a much ⁇ ⁇ Mgher-density- an-standard-si-zed-eom- ⁇ plant-s ⁇ -and-during a-time-of year-when other crops are not grown.
  • Other "baby” vegetables can also be grown in this way, such as baby turnips, eggplants, and canots.
  • Another embodiment of the invention is a method of producing a transgenic plant that is able to grown at a higher altitude or in a cooler temperature region that a conesponding non-transgenic plant, comprising introducing into the plant an isolated nucleic acid that enhances BONl, BON2, BON3, BAPl and/or BAL in the plant.
  • the invention also encompases the plant produced by the method, where the plant are able to be grown at a higher altitude or in a cooler temperature region that a conesponding non-transgenic plant.
  • Another embodiment of the invention is the production of larger plants, or plants that grow at lower than normal temperatures or to modulate homeostasis at higher altitudes.
  • Such plants can be produced by enhancing BONl, BON2, BON3 or BAPl, e.g., by overexpression as described herein.
  • Such larger plants can be used in situations where larger fruits or vegetables are desired, or where increased biomass (total amount of vegetable matter produced by the plant per unit of land) is desired, e.g., for biomass for paper production.
  • a 0.5 kb genomic fragment outside the left border of the T-DNA was rescued by using Universal Genome Walker kit (Clontech, Palo Alto, California, USA).
  • the sequence of this 0.5 kb fragment revealed its location on TAC clone K22G18 from the Arabidopsis Biological Resource Center.
  • a 6.5 kb BamHL fragment containing the gene was subcloned from this TAC clone.
  • a cDNA clone was isolated from an Arabidopsis cDNA library CD4-14 obtained from the Arabidopsis Biological Resource Center.
  • BON1-GUS fusion a 5 kb BamHL/BglLL fragment was cloned into PZP212 vector (Diener, A.C., H. Li et al. (2000) Plant Cell 12(6):853-70).
  • aBam ⁇ LL site was introduced by PCR method to the genomic BONl gene to replace the stop codon.
  • the 3xHA epitope was amplified and ligated into the BamHL site at the last codon of BONl so that it would be in frame with BONl .
  • the BONl -HA fusion was then inserted into pCGN-NOS .
  • Plasmids were introduced into Agrobacterium strain ASE (Fraley, R.T., S.G. Rogers et al. (1985) Bio/Technology 3:629-635) or GV3101 (Koncz, C, J. Schell (1986) Mol. Gen. Genet. 204:383-396) by electroporation, and transformed into wild-type Col-0, bonl-l/+ or bonl-1 by the floral dip method (Clough, SJ. and A.F. Bent (1998) Plant J. 16(6):735-43).
  • Leaf mesophyll protoplasts were isolated from 3-week-old Arabidopsis plants and were transfected by a modified polyethylene glycol method as described previously (Abel, S. and A. Theologis (1994) Plant J. 5(3):421-7). 10,000 protoplasts were transfected with 10 ⁇ g of DNA and then incubated at 23°C for 9 hours before subcelluar localization was determined with a confocal microscope. Confocal laser scanning micrographs of transfected protoplasts were recorded using a Leica microscope (Leica Microsystems, Heidelburg, Germany) equipped with a laser scanning unit (TCS NT).
  • TCS NT laser scanning unit
  • Two hybrid screen DNA was prepared from the Arabidopsis thaliana MATCFfMAKER cDNA library in E. coli (Clontech, Palo Alto, California, USA).
  • the library complexity was 3xl0 6 .
  • DNA was transformed into yeast strain PJ69-4 (James, P., J. Halladay et al (1996) Genetics 144(4): 1425-36) and 2.5xl0 7 yeast transformants were pooled as the library.
  • the bait (A domain of BONl) was cloned between EcoRI and BamHL sites of pGBD-C2 and transformed into PJ49-4a.
  • the library cells were mated with the bait A cells.
  • a screening protocol Robottson, L.S., H.C.
  • BONl is a gene required for nomial plant size at 22°C.
  • the inflorescence stem is thinner and shorter than the- wild type (Fig. ID).
  • Fig. ID The bonl-1 plants nevertheless develop relatively normal flowers and siliques and are completely fertile, bonl-1 does not have a dramatic effect on the timing of developmental process. It bolts and sets seeds only slightly earlier than the wild type.
  • the bonl phenotype is genetically distinct from that of previously identified hormonal dwarf mutants such as axrl, det2, or Ctrl.
  • the bonl-1 defect cannot be rescued by the addition of hormones such as gibberellin or brassinosteroid, or by mutations in hormonal signaling.
  • the bonl-1 mutant is distinct from the cold-sensitive mutants deficient in the desaturation of the lipids in the membranes (Lightner, J., D.W.J. James et al. (1994) Plant J. 6(3):401-412) because bonl-1 does not have an altered lipid composition or altered composition of saturated fatty acids in the lipids.
  • BONl is required to maintain cell size and number at 22°C.
  • the length of the bonl-1 cells is approximately seven times shorter than that of the wild type, which accounts for most of the eight-fold reduction in bonl stem length at the ⁇ n ⁇ -permissivelemperat ⁇ about two-thirds as wide as-the- wild-type cell. There are also half as many cells per epidermal circumference in a bonl-1 mutant as there are in the wild type, although the number of cells along the length of the same was nearly the same as in the wild type. Analysis of whole stem cross sections revealed similar defects in the inner cells: there were fewer and smaller inner cells inside the stem. Reduction in the size of the pavement cells was observed by SEM on the leaf epidermis.
  • BONl is expressed in Growing Tissues and the Expression is Modulated by Temperature.
  • the BONl gene was cloned on the basis of the T-DNA insertion that results in the bonl phenotype.
  • the mutation which segregated as a single recessive trait in the F 2 populations of a backcross to wild-type Columbia, was completely linked to the single T-DNA insertion in this mutant.
  • a 6.5 kb wild-type genomic fragment was isolated, which flanked the T-DNA and a conesponding cDNA of this genomic fragment. Sequence alignment of the genomic fragment and the cDNA indicates that the BONl gene is comprised of 16 exons and 15 introns (Fig. 2 A).
  • the T-DNA is inserted in the twelfth exon of the gene.
  • the bonl-1 mutant defect was complemented either with the genomic fragment or a cDNA-GFP (green fluorescent protein) fusion, confirming the identity of the gene as BONl.
  • Northern blot analysis showed that there was no wild-type RNA transcript of this gene in the mutant, bonl -2 (Fig. 2 A) was subsequently isolated in the Wassilewskijas background and it exhibited a phenotype similar to that of bonl-1 when introduced into the Columbia background.
  • the BONl gene is predicted to encode a protein of 578 amino acids.
  • the N- terminal portion of the protein consists of two calcium-dependent phospholipid- binding C2 domains (C2A and C2B) and the C-te ⁇ rrmal portion exhibits weak similarity to the A domain of integrih (Fig. 2B).
  • the BONl protein shows extensive homology to the copine gene family. Members of this family have been found in paramecium, worm, mouse, and human and are thought to be calcium-dependent phospholipid binding proteins (Creutz, C.E., J.L. Tomsig et al. (1998) J. Biol. Chem. 273(3):1393-402). In each organism there is more than one member of the gene family in the genome.
  • Arabidopsis thaliana BONl has two paralogs that have been designated BON2 and BON3. These paralogs are between 72-81% identical and 81- 91% similar to each other at the amino acid level. These plant proteins are very similar to their human counterparts. Arabidopsis BONl has 50% identity and 67% similarity to human copine I over the entire sequence, as shown in Fig. 3.
  • the level of expression of BONl RNA is regulated by temperature, especially during later development. Plants were first grown at one temperature for one month and then half of the population was moved to another temperature for 12 hours. The RNA level of BONl increased about two-fold when plants were shifted from 28°C to 22°C (Fig. 4D, lanes 1 and 2). Conversely, there was a decrease in BONl RNA level when plants were moved from 22°C to 28°C (Fig. 4D, lanes 3 and 4). In plants less than 2 weeks old, BONl had a relatively high expression level, as compared with older plants. In these plants there was not much difference in the level of BONl expression between plants grown at 22°C and those grown at 28°C.
  • BONl is a Phospholipid Binding Protein Associated With the Plasma Membrane
  • BONl contains two C2 domains at the amino-terminus.
  • C2 domains are Ca dependent phospholipid-binding domains that confer calcium and/or phospholipid modulation on the activity of the associated domain (Kopka, J., C. Pical et al. (1998) Plant Mol. Biol. 36(5):627-37).
  • the binding assay utilized a tagged (6x His) BONl protein that was expressed and purified from E. coli.
  • BONl-HA was not detected in protein extracts from chloroplast, nuclei or cell walls, but was present in the microsomal fraction.
  • the microsomal fraction of BONl-HA plants was separated on a 25% to 50% (w/v) sucrose gradient. The relative position of various membranes on the gradient was determined by assaying fractions for marker proteins specific to each membrane.
  • the BONl-HA protein exhibited a distribution similar to that of the plasma membrane ATPase (Fig. 5D), and distinct from ER, vacuole, and Golgi.
  • the plasma membrane localization of BONl indicated by cell fractionation was further supported by analysis in a transient expression system.
  • a BON1-GFP fusion under a strong promoter was electroporated into Arabidopsis protoplasts and the expression of GFP was monitored eight hours later with confocal microscope.
  • the GFP signal was mostly concentrated on the outer membrane of protoplasts, • indicating- a-plasma -membrane-localization.
  • BONl could stimulate cell expansion and cell division would be to enhance membrane trafficking by facilitating the association and fusion of vesicles with the membrane.
  • the ability of BONl to enhance vesicle aggregation in vitro was tested using an assay that measures the turbidity of a lipid solution. Aggregation of the lipid vesicles is accompanied by an increase in turbidity (monitored by the absorbance at 540 nm). Constructs encoding the full- length BONl protein, the N-terminal 2C2 domain, and the C-terminal A domain each with 6x His tags were expressed in E. coli and the resulting His-tagged proteins were purified.
  • BAPl is a BONl Interacting Protein.
  • the A domain (from Val to Pro ) was used as bait because it is the most likely segment of BONl to be involved in protein-protein interactions (Creutz, C.E., J.L. Tomsig et al. (1998) J. Biol. Chem. 273(3):1393-402).
  • a cDNA library made from the vegetative tissues of 3-week-old plants was screened for clones that signaled an interaction with this bait. Twenty-three positive clones rescue -from-the-screen are the same-gene r -which was..called-the ?O-YI Association. Proteinl ⁇ BAPl).
  • BAPl encodes a protein of 192 amino acids.
  • the two-hybrid clone comprises the entire BAPl except for the first 6 amino acids.
  • a blast search reveals that it has homology to another putative Arabidopsis protein in the database which, for purposes of this study, was called BAL:BAP1 Like.
  • SMART Simple Modular Architecture Research Tool
  • search indicates that the amino-terminal part of BAPl (approximately 120 amino acids) has sequence homology and stmctural analogy to the C2 domain.
  • C2 domain family is very divergent (Rizo, J., T.C. Sudhof (1998) J. Biol. Chem.
  • BAPl 273(26): 15879-82
  • the C2 domain of BAPl is not significantly homologous to those of BONl at the protein sequence level.
  • the C-terminal 52 amino acids of BAPl is 54% identical to those of BAL, but does not show significant homology to any known motifs.
  • BAPl Has a Similar Function to BONl.
  • BAPl is expressed ubiquitously throughout the roots, leaves, stems, and flowers with expression in leaves and stems relatively higher than in other parts of the plant, and BAPl also has higher expression in roots (Fig. 7A).
  • the expression of BAPl is modulated by temperature. There was an increase in BAPl RNA when plants were shifted from higher to lower temperature (Fig. 7B). Conversely, higher temperature repressed BAPl expression (Fig. 7B).
  • BAPl expression is affected by BONl. h the bonl-1 mutant, there was more BAPl transcript and the modulation of its expression by temperature was more pronounced than in wild type (Fig. 7B).
  • BAPl has a similar function to that of BONl
  • the BAPl gene was expressed under the CaMV 35S promoter and the transgene was introduced into the bonl-1 mutant. If the two proteins are involved in the same event, then overexpression of BAPl would likely suppress the mutant defects of the bonl mutant.
  • nine transgenic lines analyzed nine showed suppression to varying degrees, ranging from good (1), moderate (3), to weak (5) (Fig. 7C). Those plants that were partially suppressed had more elongated stems and more expanded leaves -than-the-bon-i-mutant. Thus-overexpression of PI an-suppress the defectin. bonl, but the extent of suppression may be modified by the site of transgene integration or transgene silencing.
  • the bonl phenotype differs from that of a typical cold sensitive mutation in a vital gene. Null mutations in a vital gene would be lethal at all temperatures, whereas BONl function and BONl protein are required to maintain normal plant size at low temperature. Mutants with a conditional cold-sensitive phenotype similar to that of bonl have been identified previously (Tsukaya, H., S. Naito et al. (1993) Development 118:751-764; Akamatsu, T., Y. Hanzawa et al. (1999) Plant Physiol. 121(3):715-22).
  • mutants grow normally at 28°C and have reduced stem elongation and leaf expansion at the lower temperature. It is possible that they could be involved in the same genetic pathway as BONl or in a parallel pathway that maintains size at low temperature. However, such conclusions are premature because neither the identity of the genes nor the nature of the mutations responsible for these phenotypes are known.
  • Arabidopsis mutant with increased levels of stearate, also exhibits a miniature phenotype at 22°C, which can be suppressed only at the very high temperature of 36°C (Lightner, J., D.W.J. James et al. (1994) Plant! 6(3):401-412).
  • BONl and BAPl are expressed more strongly in growing tissues than in mature tissues.
  • both BONl and BAPl expression are under temperature control in that both have elevated expression when plants are shifted from high to lower temperature.
  • BONl protein likely regulates membrane biogenesis and cell wall remodeling through exocytosis, which ultimately controls cell size.
  • All copines have a similar domain structure: the C2 amino tenninal domain and a carboxyl-terminal A domain.
  • the full length Arabidopsis BONl protein and the truncated C2 domain bind phospholipids and the binding is stimulated by calcium.
  • BONl promotes aggregation of lipid vesicles in vitro. Therefore, all copines may have similar biochemical activities in addition to structural similarities. Previous work provided only a few hints as to the subcellular localization of copines.
  • N-copine was localized to postsynaptic membranes where synaptic plasticity occurs (Nakayama, T, T T,Yaoi ⁇ et al, (-1-9-9-9) J.-Neurochem.J2(l):313 9) and one. of the.chromaffm granule-binding proteins (chromobindin 17) is likely to be a copine (Creutz, C.E., J.L. Tomsig et al. (1998) J. Biol. Chem. 273(3):1393-402).
  • the data herein show that BONl protein is mainly associated with the plasma membrane.
  • copine/BAPl proteins may be required to accelerate a process that occurs less rapidly at low temperature. Indeed, some of the mutations defective in membrane fusion are cold-sensitive in yeast (Lehman, K., G. Rossi et al. (1999) J. Cell Biol. 146(1): 125-40; Otte, S., W.J. Belden et al. (2001) J. Cell Biol. 152(3):503-518).
  • BONl functions in temperature homeostasis either by acting catalytically (increasing the fusion of vesicles with the membrane) or structurally (by associating with the plasma membrane to maintain membrane function at low temperature).
  • a protein kinase activity was attributed to the A domain of human copine HI (Caudell, E.G., jj. Caudell et al. (2000) Biochem. 39(42): 13034- 43) suggesting that these proteins have enzymatic function.
  • Copines in other organisms may function similarly to accelerate vesicle trafficking in response to specific environmental signals. For example, the neuronal copines could accelerate membrane trafficking at the synapse upon chemical stimulation thereby enhancing synaptic transmission.
  • acll, acl3 and al4 mutants are not identical to the BONl.
  • h acll mutants Tesukaya, H., S. Naito et al. (1993) Development 1-1-8 :-751-764
  • the length of -the-Gells- is less than tha of wild -type, as hx-bonl— mutants, but unlike the bonl mutants, the number of cells in the leaves and intemodes is the same as in wild type plants. In bonl, the number, as well as the size of the cells, is decreased relative to wild type plants.
  • acll mutants exhibit cessation of development of the inflorescence meristems at 22°C, whereas the meristems in bonl mutants grow shorter than wild type, but do not cease growth.
  • the acl3 and acl4 mutants were mapped to chromosomes 3 and 4, respectively (Akamatsu, T., Y. Hanzawa et al. (1999) Plant Physiol. 121(3):715-22).
  • BONl shows some similarity to GenBahk accession AB022212, which is located on Arabidopsis chromosome 5.
  • BAPl shows some similarity to GenBahk accession AL137898, which is located on Arabidopsis chromosome 3.
  • the full-length BONl gene was translationally fused with a reporter gene GUS and transformed to wild type Arabidopsis.
  • the transformants exhibited bonl-1 phenotype in that they were miniature plants with small curly leaves and short stems, very similar to bonl-1. Furthermore, this phenotype was exhibited only at 22°C, but not at 28°C, as was bonl-1. Therefore, the BON1-GUS chimeric gene acts either in a dominant negative fashion or induces cosuppression in wild type plants. This finding provides a method to generate miniature plants in other species.
  • the N-terminal 1wo-thirds of the BAPl gene was translationally fused with GUS gene and was introduced into wild-type Arabidopsis.
  • the transgenic plants exhibited dwarf phenotype and some exhibited anested growth, which led to death before flowering.
  • the phenotype is pronounced to that of the bonl-1 mutant.
  • this phenotype was temperature dependent, and was present at lower temperature, but absent at higher temperature. This finding shows that BAPl acts closely with BONl in plants. BONl and BAPl are involved in the same process of maintaining growth homeostasis at lower temperature.
  • Example 4 Overexpression of BONl.
  • the BONl gene was expressed under the control of the CaMV 35S promoter and transformed into wild type Arabidopsis, as described above. Approximately 1/5 of the transformants exhibited larger leaves and thicker stems than equivalent wild type plants, and the increase in size appeared to be due to an increase in both cell size and cell number. Overexpression of BONl therefore appears to result in increased growth and larger plants as compared to wild type.
  • the BAPl gene was expressed under the control of the CaMV 35S promoter and transformed into wild type Arabidopsis. Approximately 50% of the transformants exhibited larger leaves and more extensive root growth than equivalent wild type plants. Overexpression of BAPl therefore appears to result in increased growth and larger plants as compared to wild type.
  • Maize plants were grown in the dark until the leaves were 10 cm long. The leaves were cut into 0.5mm strips, digested in cellulase and macerozyme in a shaking flask. The protoplasts were then purified. The BONl -GFP fusion, prepared as described above, was introduced into the maize protoplasts by electroporation.
  • the chimeric protein was expressed in plasma membrane, as it was expressed in

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Abstract

La présente invention concerne des gènes et des protéines tels que BON1, BON2, BON3, BAP1 et BAL, dont l'expression module la taille d'une plante et des plantes transgéniques comprenant ces gènes et ces protéines ainsi que des procédés de production et d'utilisation de ces derniers.
PCT/US2001/020172 2000-06-23 2001-06-25 Gene bonsai, proteine de liaison de phospholipide, necessaire pour assurer la tolerance a la chaleur dans des plantes de la famille arabidopsis WO2002000697A2 (fr)

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WO2003012035A2 (fr) * 2001-07-27 2003-02-13 Icon Genetics, Inc. Usage commercial d'arabidopsis pour la production de proteines diagnostiques et therapeutiques humaines et animales
EP2298919A1 (fr) 2003-04-11 2011-03-23 CropDesign N.V. Méthode pour augmenter la résistance des plantes au stress
CN105158518A (zh) * 2015-09-24 2015-12-16 中国科学院西北高原生物研究所 一种毛癣菌扫描电镜样品的制备方法
CN112538486A (zh) * 2020-12-16 2021-03-23 河南农业大学 控制玉米株高的基因、其编码的蛋白质以及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1033405A2 (fr) * 1999-02-25 2000-09-06 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
AKAMATSU TAKU ET AL: "Expression of endoxyloglucan transferase genes in acaulis mutants of Arabidopsis." PLANT PHYSIOLOGY (ROCKVILLE), vol. 121, no. 3, November 1999 (1999-11), pages 715-721, XP002205276 ISSN: 0032-0889 cited in the application *
CREUTZ CARL E ET AL: "The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 3, 16 January 1998 (1998-01-16), pages 1393-1402, XP002205273 ISSN: 0021-9258 cited in the application *
DATABASE EMBL SEQUENCE LIBRARY [Online] 14 April 2000 (2000-04-14) BEVAN M., MURPHY G., RIDLEY P., ET AL.: " EU Arabidopsis sequencing project - Arabidopsis thaliana DNA chromosome 5, BAC clone T2I1 (ESSA project)" retrieved from HTTP://SRS6.EBI.AC.UK Database accession no. AL163912 XP002205280 & DATABASE TREMBL DATABASE [Online] 1 October 2000 (2000-10-01) BEVAN, M., ET AL. : "EU Arabidopsis sequencing project - Arabidopsis thaliana DNA chromosome 5, BAC clone T2I1 (ESSA project)" Database accession no. Q9LY30 *
DATABASE EMBL SEQUENCE LIBRARY [Online] 15 January 1999 (1999-01-15) SATO S., NAKAMURA Y., ET AL.: "Structural analysis of Arabidopsis thaliana chromosome 5. X. Sequence features of the regions of 3,076,755 bp covered by sixty P1 and TAC clones - Arabidopsis thaliana genomic DNA, chromosome 5, TAC clone:K22G18" retrieved from HTTP://WWW.EBI.AC.UK/CGI-BIN/EMBLFETCH Database accession no. AB022212 XP002205279 cited in the application & DATABASE TREMBL DATABASE [Online] 1 March 2001 (2001-03-01) SATO, S., ET AL. : "Copine-like protein" retrieved from HTTP://SRS6.EBI.AC.UK/SRS6BIN/CGI-BIN/WGET Z?-E+ÄSWALL-ID:Q9FH53] Database accession no. Q9FH53 *
DATABASE EMBL SEQUENCE LIBRARY [Online] 16 June 2000 (2000-06-16) NAKAMURA Y., ASAMIZU E.: " Arabidopsis thaliana cDNA clone:SQ064d10F, 3' end - A large scale analysis of cDNA in Arabidopsis thaliana: Generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries"" retrieved from HTTP://SRS6.EBI.AC.UK/SRS6BIN/CGI-BIN/WGET Z?-E+ÄEMBL-ID:AV557270] Database accession no. AV557270 XP002205278 *
DATABASE EMBL SEQUENCE LIBRARY [Online] 31 January 2000 (2000-01-31) DE HAAN M., MAARSE A.C., GRIVELL L.A., ET AL. : "Arabidopsis thaliana DNA chromosome 3, BAC clone T20K12, EU Arabidopsis sequencing project" retrieved from HTTP://SRS6.EBI.AC.UK/SRS6BIN/CGI-BIN/WGET Z?-E+ÄEMBL-ID:ATT20K12] Database accession no. AL137898 XP002205281 & DATABASE TREMBL DATABASE [Online] 1 October 2000 (2000-10-01) DE HAAN, M., ET AL. : "EU Arabidopsis thaliana sequencing project - cross reference to EMBL-entry AL137898" retrieved from HTTP://SRS6.EBI.AC.UK/SRS6BIN/CGI-BIN/WGET Z?-E+ÄSWALL-ID:Q9M2E5] Database accession no. Q9M2E5 *
DATABASE EMBL SEQUENCE LIBRARY [Online] 9 September 1999 (1999-09-09) CHEN J., MOMIYAMA M., ET AL.: "Arabidopsis thaliana Gene Expression MicroArray - 701553389 A. thaliana, Columbia Col-0, root-2 Arabidopsis thaliana cDNA clone 701553389, mRNA sequence" retrieved from HTTP://SRS6.EBI.AC.UK/SRS6BIN/CGI-BIN/WGET Z?-E+ÄEMBL-ID:AI997331] Database accession no. AI997331 XP002205282 *
HUA JIAN ET AL: "Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genes." GENES & DEVELOPMENT, vol. 15, no. 17, 1 September 2001 (2001-09-01), pages 2263-2272, XP001074049 ISSN: 0890-9369 *
KOPKA JOACHIM ET AL: "Ca2+/phospholipid-binding (C2) domain in multiple plant proteins: Novel components of the calcium-sensing apparatus." PLANT MOLECULAR BIOLOGY, vol. 36, no. 5, March 1998 (1998-03), pages 627-637, XP002205274 ISSN: 0167-4412 cited in the application *
LIGHTNER J ET AL: "A MUTANT OF ARABIDOPSIS WITH INCREASED LEVELS OF STEARIC ACID" PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 106, 1994, pages 1443-1451, XP002034186 ISSN: 0032-0889 cited in the application *
TSUKAYA HIROKAZU ET AL: "A new class of mutations in Arabidopsis thaliana, acaulis 1, affecting the development of both inflorescences and leaves." DEVELOPMENT (CAMBRIDGE), vol. 118, no. 3, 1993, pages 751-764, XP002205275 ISSN: 0950-1991 cited in the application *
WEIGEL DETLEF ET AL: "Activation tagging in Arabidopsis" PLANT PHYSIOLOGY, AMERICAN SOCIETY OF PLANT PHYSIOLOGISTS, ROCKVILLE, MD, US, vol. 122, no. 4, April 2000 (2000-04), pages 1003-1013, XP002185435 ISSN: 0032-0889 cited in the application *

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WO2003012035A2 (fr) * 2001-07-27 2003-02-13 Icon Genetics, Inc. Usage commercial d'arabidopsis pour la production de proteines diagnostiques et therapeutiques humaines et animales
WO2003012035A3 (fr) * 2001-07-27 2005-05-19 Icon Genetics Inc Usage commercial d'arabidopsis pour la production de proteines diagnostiques et therapeutiques humaines et animales
EP2298919A1 (fr) 2003-04-11 2011-03-23 CropDesign N.V. Méthode pour augmenter la résistance des plantes au stress
EP2311964A1 (fr) 2003-04-11 2011-04-20 CropDesign N.V. Method pour augmenter la resitance des plantes au stress
CN105158518A (zh) * 2015-09-24 2015-12-16 中国科学院西北高原生物研究所 一种毛癣菌扫描电镜样品的制备方法
CN112538486A (zh) * 2020-12-16 2021-03-23 河南农业大学 控制玉米株高的基因、其编码的蛋白质以及应用
CN112538486B (zh) * 2020-12-16 2022-02-15 河南农业大学 控制玉米株高的基因、其编码的蛋白质以及应用

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